Compositions and methods for generating and characterizing recombinant antigen binding molecules

Abstract

The present disclosure relates generally to the field of immunology, and particularly relates to compositions, methods, and systems for the analysis and generation of antigen-binding molecules produced by immune cells obtained from tissue samples (e.g., antibodies produced by B cells in tumor tissue samples or TCRs produced by T cells in tumor tissue samples) using spatial methodologies, and for the production and characterization of recombinant antigen-binding molecules (e.g., antibodies, TCRs) with desired properties.

What is claimed is: 
     
         1 . A method for identifying a tumor-specific antigen-binding molecule (ABM), the method comprising:
 a) providing a tissue sample comprising one or more cells expressing an ABM;   b) attaching an analyte of an ABM-expressing cell of the tissue sample to a capture domain of a first capture probe of a substrate comprising an array of capture probes attached thereto, the first capture probe comprising (i) a spatial barcode sequence and (ii) the capture domain, the capture domain comprising a capture sequence, wherein the analyte of the ABM-expressing cell comprises a sequence or portion of a sequence encoding the ABM expressed by the ABM-expressing cell or a reverse complement thereof,   c) using the analyte of the ABM-expressing cell and the first capture probe attached thereto to generate a spatially barcoded polynucleotide comprising (i) all or part of a sequence of the analyte of the ABM-expressing cell or a reverse complement thereof and (ii) the spatial barcode sequence or a reverse complement thereof, and   d) determining all or a part of the nucleic acid sequences of (i) and (ii);   e) using the determined nucleic acid sequence of the analyte from (c) to produce a recombinant ABM;   f) coupling the recombinant ABM to a reporter oligonucleotide comprising a reporter barcode sequence, thereby generating a barcoded recombinant ABM; and   g) contacting the barcoded recombinant ABM with a tumor sample, and identifying the recombinant ABM as an ABM having specificity for the tumor sample if the barcoded recombinant antibody is capable of binding to an antigen associated with the tumor sample, optionally wherein the tumor sample is a tissue sample.   
     
     
         2 . The method of  claim 1 , wherein the ABM expressed by the ABM-expressing cell is an immune cell receptor. 
     
     
         3 . The method of  claim 2 , wherein the immune cell receptor is a BCR or a TCR. 
     
     
         4 . The method of any one of  claims 1-3 , wherein the ABM expressed by the ABM-expressing cell is a secreted antibody. 
     
     
         5 . The method of any one of  claims 1-4 , wherein the capture sequence of the capture domain is a homopolymeric sequence. 
     
     
         6 . The method of any one of  claims 1-5 , wherein the capture sequence of the capture domain is a defined non-homopolymeric sequence. 
     
     
         7 . The method of  claim 6 , wherein the defined non-homopolymeric sequence is a sequence that binds to the analyte. 
     
     
         8 . The method of  claim 6 , wherein the defined non-homopolymeric sequence specifically binds to a nucleic acid sequence encoding a region of the ABM. 
     
     
         9 . The method of any one of  claims 1-8 , wherein the ABM is selected from: a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, an immunoglobulin kappa light chain, an immunoglobulin lambda light chain, an immunoglobulin heavy chain, or a combination thereof. 
     
     
         10 . The method of  claim 8 or 9 , wherein the region of the ABM is a constant region of the ABM or a variable region of the ABM. 
     
     
         11 . The method of  claim 5 , wherein the homopolymeric sequence is a poly(T) sequence. 
     
     
         12 . The method of any one of  claims 1-11 , further comprising:
 sequencing the spatially barcoded polynucleotide.   
     
     
         13 . The method of any one of  claims 1-12 , wherein the tissue sample is mounted on the substrate comprising the array of capture probes during (a) or (b). 
     
     
         14 . The method of  claim 13 , wherein a portion of the tissue sample comprising the ABM-expressing cell is in contact with the first capture probe of the array of capture probes. 
     
     
         15 . The method of any one of  claims 1-14 , wherein the method comprises, following (a):
 releasing the analyte from the ABM-expressing cell of the tissue sample; and   optionally migrating the analyte to the substrate comprising the array of capture probes attached thereto.   
     
     
         16 . The method of any one of  claims 1-12 or 15 , wherein the substrate comprising the array of capture probes attached thereto is a second substrate, wherein the tissue sample is mounted on a first substrate during (a) or (b), and optionally, wherein the method comprises releasing the analyte from the ABM-expressing cell of the tissue sample; and optionally migrating the analyte to the second substrate comprising the array of capture probes attached thereto. 
     
     
         17 . The method of any one of  claims 1-16 , wherein the analyte is a nucleic acid analyte. 
     
     
         18 . The method of any one of  claims 1-17 , wherein the analyte comprises a sequence encoding a variable region and/or a constant region of the ABM. 
     
     
         19 . The method of  claim 18 , wherein the variable region comprises a VJ or a VDJ sequence. 
     
     
         20 . The method of  claim 18 or 19 , further comprising amplifying the spatially barcoded polynucleotide to generate a spatially barcoded double-stranded nucleic acid library member comprising the sequence encoding the variable region and the constant region of the ABM. 
     
     
         21 . The method of  claim 20 , further comprising removing all or a portion of the sequence encoding the constant region of the ABM from the spatially barcoded double-stranded nucleic acid library member or amplicon thereof. 
     
     
         22 . A method for identifying a tumor-specific antibody, the method comprising:
 a) providing a first tumor tissue sample comprising one or more cells expressing one or more antibodies;   b) determining all or a part of the nucleic acid sequences encoding the one or more antibodies;   c) using the determined nucleic acid sequences to produce a recombinant antibody;   d) coupling the recombinant antibody to a reporter oligonucleotide comprising a reporter barcode sequence, thereby generating a barcoded recombinant antibody; and   e) contacting the barcoded recombinant antibody with a second tumor sample, and identifying the recombinant antibody as an antibody having specificity for the second tumor sample if the barcoded recombinant antibody is capable of binding to an antigen associated with the second tumor sample.   
     
     
         23 . The method of  claim 22 , wherein the first tumor tissue sample and/or the second tumor sample is derived from a solid tumor, a soft tissue tumor, a metastatic lesion, a non-solid tumor, a circulating tumor cell (CTC) population, a tumor cell line, or a patient derived xenograft (PDX). 
     
     
         24 . The method of  claim 22 or 23 , wherein the first tumor tissue sample and the second tumor sample are derived from the same subject or a different subject. 
     
     
         25 . The method of any one of  claims 22-24 , wherein the first tumor tissue sample and the second tumor sample are derived from the same tumor. 
     
     
         26 . The method of any one of  claims 22-25 , wherein step (e) further comprises contacting the barcoded recombinant antibody with a control tissue sample. 
     
     
         27 . The method of  claim 26 , wherein the control sample is (i) a non-tumor tissue sample or (ii) a tissue sample that the barcoded recombinant antibody is not expected to bind. 
     
     
         28 . The method of any one of  claims 22-27 , wherein the method further comprises contacting the second tumor sample with a composition comprising one or more of the following:
 i) one or more barcoded immune-cell marker antibodies and/or barcoded tumor-cell marker antibodies;   ii) one or more barcoded therapeutic antibodies; and   iii) the barcoded recombinant antibody identified as having specificity for the second tumor sample.   
     
     
         29 . The method of  claim 28 , wherein the one or more therapeutic antibodies is selected from the group consisting of abciximab, abciximab, adalimumab, aducanumab, alacizumab, alemtuzumab, alirocumab, alirocumab, ascrinvacumab, atezolizumab, atinumab, bapineuzumab, basiliximab, basiliximab, belimumab, bevacizumab, blinatumomab, blosozumab, bococizumab, brentuximab, canakinumab, caplacizumab, capromab, certolizumab, cetuximab, crenezumab, daclizumab, daratumumab, demcizumab, denosumab, denosumab, dinutuximab, ecukinumab, eculizumab, eculizumab, efalizumab, elotuzumab, enoticumab, etaracizumab, evinacumab, evolocumab, evolocumab, fasinumab, fulranumab, gantenerumab, golimumab, ibritumomab, icrucumab, idarucizumab, idarucizumab, inciacumab, infliximab, ipilimumab, mepolizumab, natalizumab, necitumumab, nesvacumab, nivolumab, obinutuzumab, ofatumumab, omalizumab, opicinumab, orticumab, ozanezumab, palivizumab, palivizumab, panitumumab, pembrolizumab, pertuzumab, ponezumab, ralpancizumab, ramucirumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, rinucumab, rituximab, romosozumab, siltuximab, solanezumab, stamulumab, tadocizumab, tanezumab, tocilizumab, trastuzumab, ustekinumab, vedolizumab, and vesencumab. 
     
     
         30 . The method of any one of  claims 28-29 , wherein the one or more immune-cell marker antibodies is selected from the group consisting of antibodies having specificity for one or more of B cells, T cells, monocytes, macrophages, granulocytes (basophil, eosinophil, neutrophil), dendritic cells, NK cells, and NKT cells. 
     
     
         31 . The method of any one of  claims 28-30 , wherein the one or more tumor-cell marker antibodies is selected from the group consisting of antibodies having specificity for ALK, alpha-fetoprotein (AFP), beta-2-microglobulin (B2M), beta-human chorionic gonadotropin (Beta-hCG), bladder tumor antigen (BTA), BRCA1, BRCA2, BCR-ABL fusion gene (Philadelphia chromosome), BRAF V600 mutations, C-kit/CD117, CA15-3/CA27.29, CA-125, CA 27.29, carcinoembryonic antigen (CEA), CD20, CD22, CD25, CD30, CD31, CD33, CD44, CD133, CD176, CD276, estrogen receptor (ER), E-cadherin, ESPR, EGFR, EPCAM, GD2, progesterone receptor (PR), fibrin/fibrinogen, HE4 gene variants, HER2 gene variants, JAK2 gene variants, KRAS gene variants, nuclear matrix protein 22, PCA3, PML/RARα fusion gene, programmed death-ligand 1 (PD-L1 or CD274), prostate-specific antigen (PSA), TEM7, TEM8, and VEGF receptor family members. 
     
     
         32 . The method of any one of  claims 22-31 , wherein identifying the recombinant antibody as an antibody having specificity for the second tumor sample further comprises quantifying levels of gene expression and protein marker expression in the second tumor sample. 
     
     
         33 . The method of  claim 32 , further comprising using the quantified levels for identification of biomarkers specific for the second tumor sample and/or a subject from whom the second tumor sample is obtained. 
     
     
         34 . The method of any one of  claims 22-33 , further comprising quantifying binding affinity of one or more therapeutic antibodies to the second tumor sample. 
     
     
         35 . The method of  claim 34 , further comprising using the quantified binding affinity as an indicator of efficacy of treating a tumor with the one or more therapeutic antibodies. 
     
     
         36 . The method of any one of  claims 34-35 , further comprising using the quantified binding affinity to monitor antigen escape of a tumor from the one or more therapeutic antibodies over time. 
     
     
         37 . The method of any one of  claims 23-36 , wherein identifying the recombinant antibody as an antibody having specificity for the second tumor sample further comprises comparing the determined nucleic acid sequences encoding the antibody to a genomic DNA sequence from the second tumor sample to confirm antigen specificity of the antibody. 
     
     
         38 . The method of  claim 37 , wherein the genomic DNA sequence is obtained from a single cell in the second tumor sample. 
     
     
         39 . The method of  claim 37 , wherein the genomic DNA sequence is obtained from a plurality of cells in the second tumor sample. 
     
     
         40 . The method of any one of  claims 37-39 , wherein the genomic DNA sequence is obtained by whole-genome sequencing. 
     
     
         41 . The method of any one of  claims 22-40 , wherein identifying the recombinant antibody as an antibody having specificity for the second tumor sample further comprises comparing the determined nucleic acid sequences encoding the antibody to a sequence of a ribonucleic acid (RNA) molecule from the second tumor sample to confirm antigen specificity of the antibody. 
     
     
         42 . The method of  claim 41 , wherein the RNA molecule is obtained from a single cell in the second tumor sample. 
     
     
         43 . The method of  claim 41 , wherein the RNA molecule is obtained from a plurality of cells in the second tumor sample. 
     
     
         44 . The method of any one of  claims 41-43 , further comprising obtaining the sequence of the RNA molecule. 
     
     
         45 . The method of any one of  claims 22-44 , further comprising determining a nucleic acid sequence of a messenger RNA (mRNA) from a single B cell and/or from a single tumor cell within the first tumor tissue sample or the second tumor sample. 
     
     
         46 . The method of  claim 45 , wherein the determining comprises binding one or more nucleic acid barcode molecules to the mRNA and optionally generating a complementary DNA (cDNA) via reverse transcription. 
     
     
         47 . The method of  claim 46 , wherein the one or more nucleic acid barcode molecules independently comprise one or more barcode sequences. 
     
     
         48 . The method of  claim 47 , wherein the one or more barcode sequences is selected from the group consisting of a sample barcode, a tissue barcode, a cell barcode, a spatial barcode, and a unique molecular identifier (UMI). 
     
     
         49 . The method of any one of  claims 46-48 , wherein the one or more nucleic acid barcode molecules are coupled to a microcapsule. 
     
     
         50 . The method of  claim 49 , wherein the microcapsule comprises a bead. 
     
     
         51 . The method of any one of  claims 45-50 , wherein the determining includes whole transcriptome sequencing. 
     
     
         52 . The method of any one of  claims 45-51 , wherein the determining comprises next-generation sequencing (NGS). 
     
     
         53 . The method of any one of  claims 22-52 , further comprising generating a chimeric antigen receptor (CAR) using the nucleic acid sequence of the recombinant antibody. 
     
     
         54 . The method of any one of  claims 22-53 , further comprising administering a composition comprising the recombinant antibody or a fragment thereof to a subject in need thereof. 
     
     
         55 . The method of any one of  claims 23-54 , further comprising administering a composition comprising an immune cell expressing the recombinant antibody or a fragment thereof to a subject in need thereof. 
     
     
         56 . The method of any one of  claims 22-55 , further comprising comparing the determined nucleic acid sequence of the recombinant antibody to sequences of known antibodies in order to identify the antibody as a tumor-specific antibody. 
     
     
         57 . The method of any one of  claims 22-56 , further comprising using a filter that takes into account clonal expansions to identify the recombinant antibody as a tumor-specific antibody. 
     
     
         58 . The method of any one of  claims 22-57 , further comprising using a filter that takes into account gene expression profiles of B cells to identify the recombinant antibody as a tumor-specific antibody. 
     
     
         59 . The method of any one of  claims 22-58 , further comprising using a filter that takes into account somatic hypermutation and isotype usage to identify the recombinant antibody as a tumor-specific antibody. 
     
     
         60 . A method for generating a recombinant antibody, the method comprising:
 a) providing a first tumor tissue sample;   b) determining all or a part of the nucleic acid sequences encoding one or more antibodies produced by one or more cells of the first tumor tissue sample; and   c) using the determined nucleic acid sequences to produce a recombinant antibody.   
     
     
         61 . The method of  claim 60 , further comprising coupling a reporter oligonucleotide comprising a reporter barcode sequence to the recombinant antibody to generate a barcoded recombinant antibody. 
     
     
         62 . The method of  claim 60 or 61 , wherein the reporter barcode sequence of the reporter oligonucleotide comprises one or more unique identifiers for the recombinant antibody. 
     
     
         63 . The method  claim 62 , further comprising determining all or a part of the nucleic acid sequence of the one or more unique identifiers to identify the barcoded recombinant antibody. 
     
     
         64 . The method of any one of  claims 60-63 , wherein the reporter oligonucleotide comprises an adapter region that allows for downstream analysis of the recombinant antibody. 
     
     
         65 . The method of  claim 64 , wherein the adapter region comprises a primer binding site and/or a cleavage site. 
     
     
         66 . The method of any one of  claims 60-65 , further comprising contacting the barcoded recombinant antibody to a tumor cell obtained from a second tumor tissue sample. 
     
     
         67 . The method of  claim 66 , further comprising identifying the recombinant antibody as an antibody having specificity for the second tumor tissue sample if the barcoded recombinant antibody is capable of binding to an antigen associated with the second tumor tissue sample. 
     
     
         68 . The method of  claim 66 or 67 , wherein the first and/or the second tumor tissue sample is derived from a solid tumor, a soft tissue tumor, a metastatic lesion, a non-solid tumor, a circulating tumor cell (CTC) population, a tumor cell line, or a patient derived xenograft (PDX). 
     
     
         69 . The method of any one of  claims 66-68 , wherein the first and the second tumor tissue samples are derived from the same subject or a different subject. 
     
     
         70 . The method of any one of  claims 66-68 , wherein the first and the second tumor tissue samples are derived from the same tumor. 
     
     
         71 . The method of any one of  claims 66-70 , wherein the method comprises contacting the second tumor tissue sample with a composition comprising one or more of the following:
 i) one or more barcoded immune-cell marker antibodies and/or barcoded tumor-cell marker antibodies;   ii) one or more barcoded therapeutic antibodies; and   iii) the barcoded recombinant antibody identified as having specificity for the second tumor tissue sample.   
     
     
         72 . The method of  claim 71 , wherein the one or more therapeutic antibodies is selected from the group consisting of abciximab, abciximab, adalimumab, aducanumab, alacizumab, alemtuzumab, alirocumab, alirocumab, ascrinvacumab, atezolizumab, atinumab, bapineuzumab, basiliximab, basiliximab, belimumab, bevacizumab, blinatumomab, blosozumab, bococizumab, brentuximab, canakinumab, caplacizumab, capromab, certolizumab, cetuximab, crenezumab, daclizumab, daratumumab, demcizumab, denosumab, denosumab, dinutuximab, ecukinumab, eculizumab, eculizumab, efalizumab, elotuzumab, enoticumab, etaracizumab, evinacumab, evolocumab, evolocumab, fasinumab, fulranumab, gantenerumab, golimumab, ibritumomab, icrucumab, idarucizumab, idarucizumab, inciacumab, infliximab, ipilimumab, mepolizumab, natalizumab, necitumumab, nesvacumab, nivolumab, obinutuzumab, ofatumumab, omalizumab, opicinumab, orticumab, ozanezumab, palivizumab, palivizumab, panitumumab, pembrolizumab, pertuzumab, ponezumab, ralpancizumab, ramucirumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, rinucumab, rituximab, romosozumab, siltuximab, solanezumab, stamulumab, tadocizumab, tanezumab, tocilizumab, trastuzumab, ustekinumab, vedolizumab, and vesencumab. 
     
     
         73 . The method of  claim 71 or 72 , wherein the one or more immune-cell marker antibodies is selected from the group consisting of antibodies having specificity for one or more of B cells, T cells, monocytes, macrophages, granulocytes (basophil, eosinophil, neutrophil), dendritic cells, NK cells, and NKT cells. 
     
     
         74 . The method of any one of  claims 71-73 , wherein the one or more tumor-cell marker antibodies is selected from the group consisting of antibodies having specificity for ALK, alpha-fetoprotein (AFP), beta-2-microglobulin (B2M), beta-human chorionic gonadotropin (Beta-hCG), bladder tumor antigen (BTA), BRCA1, BRCA2, BCR-ABL fusion gene (Philadelphia chromosome), BRAF V600 mutations, C-kit/CD117, CA15-3/CA27.29, CA-125, CA 27.29, carcinoembryonic antigen (CEA), CD20, CD22, CD25, CD30, CD31, CD33, CD44, CD133, CD176, CD276, estrogen receptor (ER), E-cadherin, ESPR, EGFR, EPCAM, GD2, progesterone receptor (PR), fibrin/fibrinogen, HE4 gene variants, HER2 gene variants, JAK2 gene variants, KRAS gene variants, nuclear matrix protein 22, PCA3, PML/RARα fusion gene, programmed death-ligand 1 (PD-L1 or CD274), prostate-specific antigen (PSA), TEM7, TEM8, and VEGF receptor family members. 
     
     
         75 . A recombinant antibody or a functional fragment thereof generated or identified by a method according to any one of  claims 22 to 74 . 
     
     
         76 . A recombinant nucleic acid comprising a nucleic acid sequence that encodes the recombinant antibody or functional fragment thereof of  claim 75 . 
     
     
         77 . The recombinant nucleic acid of  claim 76 , wherein the recombinant nucleic acid is further configured as an expression cassette in a vector. 
     
     
         78 . The recombinant nucleic acid of  claim 77 , wherein the vector is a plasmid vector or a viral vector. 
     
     
         79 . A recombinant cell comprising a recombinant nucleic acid according to any one of  claims 75-78 . 
     
     
         80 . The recombinant cell of  claim 79 , wherein the recombinant cell is a prokaryotic cell or a eukaryotic cell. 
     
     
         81 . A composition comprising a pharmaceutically acceptable excipient and one or more of the following:
 a) a recombinant antibody or the functional fragment thereof of  claim 75 ;   b) a recombinant nucleic acid according to any one of  claims 76 to 78 ; or   c) a recombinant cell according to claim  79  or  80 .   
     
     
         82 . A composition comprising one or more of the following:
 a) one or more barcoded immune-cell marker antibodies and/or barcoded tumor-cell marker antibodies;   b) one or more barcoded therapeutic antibodies; or   c) the recombinant antibody identified in  claim 75  as having specificity for the second tumor sample, wherein the recombinant antibody is barcoded.   
     
     
         83 . A kit comprising one or more of the following:
 a) the recombinant antibody or the functional fragment thereof of  claim 75 ;   b) a recombinant nucleic acid according to any one of  claims 76 to 78 ; or   c) a recombinant cell according to claim  79  or  80 ; or   instructions for use thereof.   
     
     
         84 . A method for characterizing antibody specificity or target specificity, the method comprising:
 a) providing a first tumor tissue sample;   b) determining all or a part of one or more nucleic acid sequences encoding one or more antibodies produced by B cells in the tumor tissue sample;   c) using the determined nucleic acid sequences to produce one or more recombinant antibodies;   d) coupling the one or more recombinant antibodies to a reporter oligonucleotide comprising a reporter barcode sequence to generate one or more barcoded recombinant antibodies;   e) contacting the one or more barcoded recombinant antibodies with a second tumor sample, and identifying the one or more recombinant antibodies as having specificity for the tumor sample if the one or more barcoded recombinant antibodies is capable of binding to an antigen associated with the second tumor sample, optionally wherein the second tumor sample is a tissue sample; and   f) analyzing RNA expression and protein marker expression for the first tumor tissue samples and/or second tumor sample to determine the one or more recombinant antibody's specificity and target specificity.   
     
     
         85 . A method for enhanced identification of patient-specific or population-specific biomarkers, the method comprising:
 a) providing a tumor tissue sample comprising a plurality of B cells;   b) determining all or a part of the nucleic acid sequences encoding one or more antibodies produced by B cells in the tumor tissue sample;   c) using the determined nucleic acid sequences to produce a recombinant antibody;   d) coupling the recombinant antibody to a reporter oligonucleotide comprising a reporter barcode sequence to generate a barcoded recombinant antibody;   e) contacting the barcoded recombinant antibody with a second tumor sample, and identifying the recombinant antibody as an antibody having specificity for the second tumor sample if the barcoded recombinant antibody is capable of binding to an antigen associated with the second tumor sample, optionally wherein the second tumor sample is a tissue sample; and   f) analyzing RNA expression and protein marker expression for the second tumor sample to identify one or more biomarkers specific for the second tumor sample or for a population of tumor tissue samples.   
     
     
         86 . A method for monitoring antigen escape in an individual who has been treated with an antibody-based therapy, the method comprising:
 a) providing a tumor tissue sample comprising a plurality of B cells;   b) determining all or a part of the nucleic acid sequences encoding one or more antibodies produced by the B cells in the tumor tissue sample;   c) using the determined nucleic acid sequences to produce a recombinant antibody;   d) coupling the recombinant antibody to a reporter oligonucleotide comprising a reporter barcode sequence to generate a barcoded recombinant antibody;   e) contacting the barcoded recombinant antibody with a second tumor tissue sample, and identifying the recombinant antibody as an antibody having specificity for the tumor tissue sample if the barcoded recombinant antibody is capable of binding to an antigen associated with the second tumor tissue sample;   f) quantifying binding affinity of a barcoded therapeutic antibody to the second tumor tissue sample, wherein the quantified binding affinity is indicative of the therapeutic antibody's efficacy in treating the tumor; and   g) optionally using the quantified binding affinity to monitor antigen escape from the therapeutic antibody over time.   
     
     
         87 . A method for characterizing a potential antigen for an antibody or fragment thereof, the method comprising:
 a) providing a tumor tissue sample comprising a plurality of B cells;   b) determining all or a part of the nucleic acid sequences encoding one or more antibodies produced by the B cells in the tumor tissue sample;   c) using the determined nucleic acid sequences to produce a recombinant antibody;   d) coupling the recombinant antibody to a reporter oligonucleotide comprising a reporter barcode sequence to generate a barcoded recombinant antibody;   e) contacting the barcoded recombinant antibody with a second tumor tissue sample, and identifying the recombinant antibody as an antibody having specificity for the tumor tissue sample if the barcoded recombinant antibody is capable of binding to an antigen associated with the second tumor tissue sample; and   f) quantifying binding affinity of the one or more antibodies to the second tumor tissue sample, and using the quantified binding affinity to determine if the one or more antibodies compete with one another for binding to the second tumor tissue sample; and   g) optionally co-associating the quantified binding affinity with RNA expression analysis to identify potential antigen.   
     
     
         88 . A method for identifying a tumor-specific antibody, comprising:
 a) contacting a barcoded recombinant antibody with a tumor tissue sample; and   b) identifying the barcoded recombinant antibody as a tumor-specific antibody if the barcoded recombinant antibody is capable of binding to an antigen associated with the tumor tissue sample,   wherein the barcoded recombinant antibody comprises a recombinant antibody coupled to a reporter oligonucleotide comprising a reporter barcode sequence, wherein the recombinant antibody is identified and/or produced by (i) providing a tumor tissue sample comprising a plurality of B cells (ii) determining all or a part of the nucleic acid sequences encoding one or more antibodies produced by the B cell cells in the tumor tissue sample, and optionally (iii) using the determined nucleic acid sequences to recombinantly produce the recombinant antibody.   
     
     
         89 . The method of  claim 88 , wherein the barcoded recombinant antibody is produced by coupling the recombinant antibody to a reporter oligonucleotide comprising a reporter barcode sequence. 
     
     
         90 . The method of any one of  claims 1-74 or 85-89 , wherein determining all or a part of the nucleic acid sequences of the analyte or all or a part of the nucleic acid sequence encoding the one or more antibodies comprises:
 a) contacting the tissue sample or tumor tissue sample with a first primer comprising a nucleic acid sequence that hybridizes to a complementary sequence in the analyte or nucleic acid sequence encoding the one or more antibodies and a functional domain;   (b) hybridizing the first primer to the analyte or the nucleic acid sequence encoding the one or more antibodies and extending the first primer using the analyte or the nucleic acid sequence encoding the one or more antibodies as a template to generate an extension product;   (c) incorporating a polynucleotide sequence comprising at least three non-templated nucleotides to the 3′ end of the extension product;   (d) hybridizing a second primer to the polynucleotide sequence comprising at least three non-templated nucleotides of the extension product of (c), wherein the second primer comprises a capture sequence;   (e) extending the extension product using the second primer as a template, thereby incorporating a complement of the capture sequence into the extension product;   (f) hybridizing the complement of the capture sequence of the extension product to a capture domain on an array, wherein the array comprises a plurality of capture probes, and wherein a capture probe of the plurality of capture probes comprises a spatial barcode and the capture domain; and   (g) determining (i) the sequence of the spatial barcode, or a complement thereof, and (ii) all or a portion of the sequence of the nucleic acid sequence, or a complement thereof, and using the determined sequences of (i) and (ii) to determine all or a part of the nucleic acid sequence of the analyte or the nucleic acid sequence encoding the one or more antibodies.   
     
     
         91 . The method of  claim 90 , wherein the first primer comprises a random sequence, optionally wherein the random sequence comprises a random hexamer or random decamer. 
     
     
         92 . The method of  claim 90 , wherein the first primer comprises a homopolymer sequence, optionally wherein the homopolymer sequence comprises a poly(T) sequence. 
     
     
         93 . The method of any one of  claims 90-92 , wherein the first primer comprises a sequence substantially complementary to a sequence in the analyte or all or a part of the nucleic acid sequence encoding the one or more antibodies encoding a constant region of an immune cell receptor, optionally wherein the immune cell receptor comprises a B cell receptor or a T cell receptor. 
     
     
         94 . The method of any one of  claims 90-93 , wherein incorporating the polynucleotide sequence to the 3′ end of the extension product in step (c) comprises the use of a terminal deoxynucleotidyl transferase or of a reverse transcriptase. 
     
     
         95 . The method of any one of  claims 90-94 , wherein the second primer comprises RNA. 
     
     
         96 . The method of any one of  claims 90-95 , wherein the method further comprises removing the analyte or the nucleic acid sequence encoding the one or more antibodies, or any other nucleic acid hybridized to the extension product, before the complement of the capture sequence of the extension product hybridizes to the capture domain of the capture probe on the array, optionally wherein the removing comprises the use of an RNase, optionally wherein the RNase is RNaseH. 
     
     
         97 . The method of any one of  claims 90-96 , wherein the method further comprises a step of extending the 3′ end of the extension product of step (e) using the capture probe as a template, thereby generating an extended capture product, and/or extending the capture probe using the extension product of step (e) as a template. 
     
     
         98 . The method of  claim 97 , wherein step (b) comprises generating one or more extension products using a plurality of primers, wherein a primer of the plurality of primers comprises a nucleic acid sequence that is substantially complementary to a sequence in the target nucleic acid and a functional domain, wherein the first primer is comprised in the plurality of primers;
 (a) hybridizing the plurality of primers to the analyte or the nucleic acid sequence encoding the one or more antibodies and extending one or more primers from the plurality of primers using the target nucleic acid as a template to generate the one or more extension products;   (b) attaching a polynucleotide sequence to the 3′ end of the one or more extension products;   (c) hybridizing the second primer to the polynucleotide sequence of the one or more extension products of (b), wherein the second primer comprises a capture sequence;   (d) extending the one or more extension products using the second primer as a template, thereby incorporating a complement of the capture sequence into the one or more extension products;   (e) hybridizing the complement of the capture sequence of the one or more extension products to a capture domain on an array, wherein the array comprises a plurality of capture probes, and wherein the capture probe of the plurality of capture probes comprises a spatial barcode and the capture domain; and   (f) determining (i) the sequence of the spatial barcode, or a complement thereof, and (ii) all or a portion of the sequence of the target nucleic acid, or a complement thereof, and using the determined sequences of (i) and (ii) to determine all or a part of the nucleic acid sequence of the analyte or the nucleic acid sequence encoding the one or more antibodies.   
     
     
         99 . The method of any one of  claims 1-74 or 85-89 , wherein determining all or a part of the nucleic acid sequence of the analyte or all or a part of the nucleic acid sequence encoding the one or more antibodies comprises:
 (a) contacting the tissue sample or tumor tissue sample with an array comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises (i) a spatial barcode and (ii) a capture domain that hybridizes to a poly(A) sequence of the analyte or the nucleic acid sequence encoding the one or more antibodies;   (b) hybridizing the capture domain to the analyte or nucleic acid sequence encoding the one or more antibodies;   (c) extending the capture probe using the analyte or nucleic acid sequence encoding the one or more antibodies as a template to generate an extended capture probe comprising a sequence encoding a CDR3, or a complement thereof, of the ABM or one or more antibodies;   (d)hybridizing one or more probes to the extended capture probe, or a complement thereof, in a portion encoding a constant region of the ABM or one or more antibodies, wherein the one or more probes comprises a binding moiety capable of binding a capture moiety;   (e) enriching the extended capture probe or the complement thereof via an interaction between the binding moiety in the one or more probes and the capture moiety; and   (f) determining (i) the sequence of the spatial barcode or a complement thereof, and (ii) all or a portion of the sequence of the analyte or nucleic acid sequence encoding the one or more antibodies, or a complement thereof, and using the determined sequences of (i) and (ii) to determine all or part of the nucleic acid sequence of the analyte or the nucleic acid sequence encoding the one or more antibodies.   
     
     
         100 . The method of  claim 99 , wherein the one or more probes hybridizes to a nucleic acid sequence encoding a constant region of the ABM or one or more antibodies, or a complement thereof. 
     
     
         101 . The method of  claim 99 or 100 , wherein the capture domain comprises a poly(T) sequence. 
     
     
         102 . The method of any one of  claims 99-101  further comprising generating the complement of the extended capture using the extended capture probe as a template, wherein the complement of the extended capture probe comprises (i) a sequence that is complementary to the spatial barcode, and (ii) a sequence that corresponds to all or a portion of the analyte or nucleic acid sequence encoding the one or more antibodies. 
     
     
         103 . The method of any one of  claims 99-102 , wherein the binding moiety comprises biotin and the capture moiety comprises streptavidin. 
     
     
         104 . The method of any one of  claims 99-103 , wherein the determining in step (f) comprises sequencing the extended capture probe or the complement thereof to determine (i) the sequence of the spatial barcode, or the complement thereof, and (ii) all or a portion of the sequence of the the analyte or nucleic acid sequence encoding the one or more antibodies, optionally wherein the sequencing comprises long read sequencing. 
     
     
         105 . The method of any one of  claims 99-104 , wherein the capture probe further comprises an adaptor domain and the method further comprises after step (e), performing a polymerase chain reaction using i) a first primer complementary to the adaptor domain of the capture probe, and ii) a second primer complementary to a portion of the analyte or nucleic acid sequence encoding the one or more antibodies in a portion encoding a variable region of the immune cell receptor of the immune cell clonotype. 
     
     
         106 . The method of  claim 105 , wherein the second primer is complementary to a nucleic acid sequence 5′ to the sequence encoding CDR3 of the ABM or the one or more antibodies. 
     
     
         107 . The method of any one of  claims 1-74 or 85-89 , wherein determining all or a part of the nucleic acid sequences of the analyte or all or a part of the nucleic acid sequence encoding the one or more antibodies comprises:
 (a) contacting the tissue sample or tumor tissue sample with an array comprising a feature, wherein the feature comprises an attached first and second probe, wherein:   a 5′ end of the first probe is attached to the feature;   the first probe comprises in a 5′ to a 3′ direction: a spatial barcode and a poly(T) capture domain, wherein the poly(T) capture domain binds specifically to the analyte or the nucleic acid sequence encoding the one or more antibodies;   a 5′ end of the second probe is attached to the feature;   a 3′ end of the second probe is reversibly blocked; and   the second probe comprises a poly(GI) capture domain;   (b) extending a 3′ end of the first probe to add a sequence that is complementary to a portion of the analyte or the nucleic acid sequence encoding the one or more antibodies;   (c) ligating an adapter to the 5′ end of analyte or the nucleic acid sequence encoding the one or more antibodies specifically bound to the first probe;   (d) adding a sequence complementary to the adapter to the 3′ end of the first probe;   (e) adding non-templated cytosines to the 3′ end of the first probe to generate a poly(C) sequence, wherein the poly(C) sequence specifically binds to the poly(GI) capture domain of the second probe;   (f) unblocking the 3′ end of the second probe and extending the 3′ end of the second probe to add a sequence comprising a sequence in the analyte or the nucleic acid sequence encoding the one or more antibodies and a sequence that is complementary to the spatial barcode;   (g) cleaving a region of the second probe at a cleavage site that is 5′ to the poly(GI) capture domain, thereby releasing the second probe from the feature; and   (h) determining (i) all or a part of the sequence of the spatial barcode, or a complement thereof, and (ii) all or a part of the sequence of the analyte or the nucleic acid sequence encoding the one or more antibodies, or a complement thereof, and using the sequences of (i) and (ii) to determine all or a part of the nucleic acid sequence of the analyte or the nucleic acid sequence encoding the one or more antibodies.   
     
     
         108 . The method of  claim 21 , wherein a single strand of the double-stranded nucleic acid library member comprises: a first adaptor, a barcode, a capture domain, a sequence of the analyte or a complement thereof, and a second adaptor. 
     
     
         109 . The method of  claim 21 , wherein removing all or a portion of the sequence encoding the constant region of the ABM from the spatially barcoded double-stranded nucleic acid library member comprises:
 (a) ligating to each end of the double-stranded member of the nucleic acid library a first restriction endonuclease recognition sequence;   (b) contacting the double-stranded member of the nucleic acid library of step (a) with a first restriction endonuclease that cleaves the first restriction endonuclease recognition sequence at each end;   (c) ligating the ends of the double-stranded member of the nucleic acid library of step (b) to generate a first double-stranded circularized nucleic acid; and   (d) amplifying the double-stranded circularized nucleic acid using a first primer and a second primer to generate a double-stranded member of the nucleic acid library lacking all, or a portion of, the analyte sequence, wherein:   the first primer comprises: (i) a sequence substantially complementary to a 3′ region of the analyte sequence, and (ii) a first functional domain comprising a sequence for attachment to a flow cell; and   the second primer comprises: (i) a sequence substantially complementary to a 5′ region of the analyte sequence, and (ii) a second functional domain comprising a primer sequence to amplify the double-stranded member of the nucleic acid library lacking all, or a portion of, encoding the constant region of the ABM.   
     
     
         110 . The method of  claim 109 , wherein the first primer comprises (i) the sequence substantially complementary to the 3′ region of the analyte sequence, and (ii) the sequence comprising the first functional domain, in 3′ to 5′ direction; and wherein the second primer comprises (i) the sequence substantially complementary to the 5′ region of the analyte sequence, and (ii) the sequence comprising the second functional domain, in a 3′ to 5′ direction. 
     
     
         111 . The method of  claim 109 or 110 , wherein ligating in step (c) is performed using a DNA ligase or using template mediated ligation. 
     
     
         112 . The method of any one of  claims 109-111 , wherein the nucleic acid library is a DNA library or a cDNA library. 
     
     
         113 . The method of any one of  claims 109-112 , wherein the method further comprises amplifying the double-stranded member of the nucleic acid library lacking all, or a portion of, the sequence encoding the constant region of the ABM using a third primer and a fourth primer, wherein:
 the third primer is substantially complementary to the first functional domain, and   the fourth primer is substantially complementary to the second functional domain.   
     
     
         114 . A method for generating a recombinant antigen-binding molecule, the method comprising:
 a) providing a tumor tissue sample comprising a plurality of immune cells;   b) determining all or a part of the nucleic acid sequences encoding one or more antigen-binding molecules expressed by the immune cells in the tumor tissue sample; and   c) using the determined nucleic acid sequences to produce a recombinant antigen-binding molecule.   
     
     
         115 . The method of  claim 92 , wherein the plurality of immune cells comprises a T cell and wherein the one or more antigen-binding molecules produced by the immune cells comprises a TCR. 
     
     
         116 . The method of  claim 16 , wherein the method comprises, following (a):
 mounting the first substrate on a first member of a support device, the first member configured to retain the first substrate;   mounting the second substrate on a second member of the support device, the second member configured to retain the second substrate;   applying a reagent medium to the first substrate and/or the second substrate, the reagent medium comprising a permeabilization agent;   operating an alignment mechanism of the support device to move the first member and/or the second member such that a portion of the tissue sample comprising the ABM-expressing cell is aligned with a portion of the array of capture probes and within a threshold distance of the array of capture probes, and such that the portion of the tissue sample and the capture probe contact the reagent medium,   wherein the permeabilization agent releases the analyte from the ABM-expressing cell.   
     
     
         117 . The method of  claim 16 , wherein the method further comprises aligning the first substrate with the second substrate, such that at least a portion of the tissue sample is aligned with at least a portion of the second substrate. 
     
     
         118 . The method of  claim 15 or 16 , wherein the migrating comprises passive migration. 
     
     
         119 . The method of  claim 15 or 16 , wherein the migrating comprises active migration, and optionally, wherein the active migration comprises electrophoresis. 
     
     
         120 . The method of  claim 17 , wherein the analyte is RNA or DNA, optionally wherein the RNA is mRNA, or the DNA is cDNA or genomic DNA. 
     
     
         121 . The method of anyone of  claims 1-21 or 116-120 , where the attaching in step (b) comprises hybridization. 
     
     
         122 . The method of any one of  claims 1-21 or 116-121 , wherein the analyte encodes V and J sequences of an immune cell receptor, preferably a BCR or TCR. 
     
     
         123 . The method of any one of  claims 1-21 or 116-121 , wherein the analyte encodes V, D, and J sequences of an immune cell receptor, preferably a BCR or TCR. 
     
     
         124 . The method of any one of  claims 1-21 or 116-121 , wherein the tissue sample is a tissue section, and optionally, wherein the tissue section is a fixed tissue section or a fresh, frozen tissue section. 
     
     
         125 . The method of  claim 124 , wherein the fixed tissue section is a formalin-fixed paraffin-embedded tissue section, a paraformaldehyde-fixed tissue section, a methanol-fixed tissue section, or an acetone-fixed tissue section. 
     
     
         126 . The method of any one of  claims 1-21 or 116-125 , wherein the tissue sample is a human sample.